Report from the visiting committee

Section des Unités de recherche Report from the visiting committee Research unit : Laboratoire Maturation des ARN et Enzymologie Moléculaire UMR 7567 University Nancy 1 February 2008

Report from the visiting committee The research unit : Name of the research unit : Laboratoire Maturation des ARN et Enzymologie Moléculaire Requested label : UMR N in case of renewal : 7567 Head of the research unit : Mrs Christiane BRANLANT University or school : Université Nancy 1 Other institutions and research organization: CNRS Date(s) of the visit : 14-15 February 2008 2

Members of the visiting committee Chairman of the commitee : Mr Alain Jacquier, Paris Other committee members : Mrs Irene Bozzoni, Rome, Italy Mr Jean-Marie Frère, Louvain, Belgique Mr Roland Marquet, Strasbourg, France Mr Vincent Nivière, Grenoble CNU, CoNRS, CSS INSERM, représentant INRA, INRIA, IRD..) representatives : Mr Daniel Locker, CoNRS representative Mr Alain Cozzone, CNU representative Observers AERES scientific representative: Mr Laszlo Tora University or school representative: M. Pierre Mutzenhard, Université Henri Poincaré Nancy Research organization representative (s) : Mr Thierry Meinnel, SDV CNRS 3

Report from the visiting committee 1 Short presentation of the research unit Number of full time researchers : 6 Number of researchers with teaching duties : 9 Number of PhD students : 15 Number of enginneers, technicians and administrative assistants : 12.6 including 7 from CNRS Numbers of HDR : 7, all are PhD students avisors Numbers of PhD students who have obtained their PhD since January 2004 : 13 Average length of a PhD during the past 4 years: 4 years Numbers PhD students currently present in the research unit 15, all of them with fellowships 15 Numbers of lab members who have been granted a PEDR : 6 Numbers of publishing lab members : 15 out of 15 2 Preparation and execution of the visit Day 1 : Welcoming and general presentation by the director of the unit on the history, the organization of the structure and its integration into the local and regional scientific environment (~ 1h 30). Overall presentation of the Molecular Enzymology team (30 min.). General presentation of the RNA Maturation team (30 min.). Division of the committee into two groups visiting and listening to presentations from each of the teams (~ 4 et 5 heures respectivement). Day 2 : Visit and presentation of the technical platforms (1 heure) Discussion with students (1 heure) Discussion with the University and CNRS observers (1 heure) Deliberation of the scientific committee in plenary session ( 3 heures) 4

3 Overall appreciation of the activity of the research unit, of its links with local, national and international partners The two senior researchers have created this lab in Nancy some 20 years ago contributing to the development of Molecular Biology in this research campus. The exceptional example of how these researchers managed to establish and expand fundamental research on Molecular Biology in Nancy must be commended. For these historical reasons, the organization of the unit is somewhat peculiar, with two big groups with very distinct research interests. Another general characteristic of the groups are their strong implication in the life of the Nancy University. The unit is comprised of a relatively large proportion of researchers with teaching duties, but even the full time researchers are quite heavily involved in teaching. Moreover, the unit hosts a relatively high number of PhD students, all with fellowships. These characteristics find their roots in the historical mission of the two senior scientists to firmly establish fundamental Molecular Biology research in Nancy. It is also worth mentioning that the senior members of the team, and in particular the head of the unit, are deeply involved in the administration of research, both locally and at the national level, and this at an exceptionally high degree, in terms of both quantitative and qualitative investments. The scientific evaluation will be described team by team, given the clear distinction between the two groups. 4 Specific appreciation team by team and/or project by project Team 1: Molecular Enzymology The molecular enzymology group studies aldehyde dehydrogenases and enzymes involved in the repair of oxidised forms of sulphur-containing amino acid residues. Their main goal is to decipher the catalytic mechanisms on the basis of biochemical, rapid kinetic methods and structural analyses. The latter are performed with the help of inhouse equipment (X-ray diffractometer) or in collaboration with other groups in Nancy (NMR) and Strasbourg (mass spectrometry). The group has recently recruited specialists in X-ray crystallography (helped by a research engineer) and in mass spectrometry and plans to recruit a specialist in protein NMR. Together with the existing expertise in protein chemistry and biochemistry (including protein engineering), this allows a very well-balanced approach to the enzyme catalysis. 1. Aldehyde dehydrogenases (ALDHs). Three enzymes have been chosen as model proteins. Their catalytic pathways involve the formation of a thioacylenzyme intermediate. For the non phosphorylating, NADPH-dependent Glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Streptococcus mutans, the pathway was found to involve an important conformation change of the cofactor after hydride transfer, which is necessary before the deacylation step can take place. The role of several residues in the catalytic pathway has been elucidated by site-directed mutagenesis as well as the slow equilibrium between free and phosphate-bound forms. A first approach of the mechanism of retinal dehydrogenase-2 has been performed by rapid kinetic methods and the rate-limiting step of the CoA-dependent methylmalonyl semialdehyde dehydrogenase characterised. For the latter enzyme, a structural explanation has been proposed for the observed properties of half-of-the-sites reactivity. 2. Repair of oxidised cysteines and methionines. Methionine sulfoxide reductases are essential in the resistance to oxidative stress. They also contain an essential Cys residue. Several classes, some of then with no sequence homologies, act according to similar mechanisms and all appear to involve a sulphurane-type intermediate which evolves into a sulphenic acid. In 3 classes, an internal disulphide bridge is subsequently formed, which is finally reduced by thioredoxin. In the 4th group, the sulphenic acid intermediate is reduced by an external system, which remains to be determined as the final reducing agent. An interesting theoretical chemistry approach on the reaction intermediates is performed in collaboration with another group in Nancy. The PilB protein of Neisseria meningitidis which contains, in addition to two different methionine reductase domains, a thioredoxin-like N-terminal domain is under study. 5

Sulfiredoxins, which reduce cysteine sulphinic into sulphenic acids are involved in the regulation of peroxiredoxin activity in eukaryotes and H2O2 cell signalling. Studies on the reaction mechanism, with a recent identification of a sulfiredoxin-peroxiredoxin complex intermediate are under progress. For all these enzymes, the group has performed pioneering and original work which has been published in excellent journals. A few years ago, oxidised forms of Met and Cys residues were usually considered as artefacts due to purification or analysis procedures. The work of the group underlines the physiological relevance of the repair mechanisms which eliminate these modified residues from functional proteins. It can be expected that their importance will be increasingly recognised in the future as well as their relationship with various stress-related pathological situations. Team 2 : RNA Maturation This team has focused its scientific activity more specifically on the definition of the role of RNP complexes in different biological processes. In all these years, the projects have been characterized by a strong scientific coherence and have benefited of the use of a large variety of powerful and modern technologies for RNP analysis. This allowed the group to acquire an internationally recognized position as leaders in the study of RNP assembly, structure and function. It is notable to remember the extensive characterization that the group has pursued on the study of U3 snrnp composition as well as in the characterization of its role in rrna processing. In particular, in the last 4 years they have succeeded in showing the existence of a new interaction between U3 snorna and the yeast pre-rrna; in addition, they have accomplished a detailed characterization of the protein components binding the different structural domains of the U3 snorna. Of relevance is also the work on the characterization of other snornps such as those belonging to the H/ACA class. The team has successfully accomplished the structure definition of the L7A protein from Archea and the analysis of its evolutionary conservation in its eukaryotic counterparts. Along the same line of research, they have also obtained the in vitro reconstitution of H/ACA RNPs active in pseudouridylation, determined, "in house", the 3D structure of NOP10-CBF5 by X-ray cristallography and defined the role of the C-terminal domains of both proteins in H/ACA srnp activity. Other relevant results have been obtained relatively to the characterization of C/D box snornp assembly factors. The results have allowed the team to make an interesting model for the C/D box biogenesis that will be very soon verified in vivo. All these studies on RNP structure and biosynthesis have led to several very good publications that have served as references for many laboratories working on similar subjects. Another research line that has yielded important contributions to the field of RNA maturation is the one on the study of HIV alternative splicing. The group has succeeded in demonstrating that the alternative choices of splice site recognition in the HIV primary transcript are determined in vitro by the differential abundance of specific SR proteins. Very detailed analyses have been carried out on the region around splice site A3 with the identification of binding sites for several hnrnps and the definition of the splicing effects determined by their competitive binding. More recently, the RNA maturation unit has been enriched by new projects aimed at defining the role of RNAprotein interactions in other cellular functions and eventually at understanding their relevance in physiological as well as in pathological conditions. A project aimed at identifying new potential functions of the Survival Motor Neuron (SMN) complex and at defining their possible correlation with the spinal muscular atrophy has been started. The project is very well planned and structured and intends to tackle the problem through the use of many different methodological approaches. The expected results will certainly add important knowledge to the field. It is also important to mention the project on Myotonic Dystrophy. The aim of the project is to define the role of the triplet expansions in sequestering specific nuclear factors and to understand the effects of such decoy on cellular splicing. The project is original and well conceived; also in this case, several different aspects of the problem are approached (RNA structure analysis, RNA-protein interaction, protein partner identification ) and the use of several different techniques is proposed. 6

Lastly, the Xist RNA biosynthesis and regulation of X-chromosome inactivation project, if confirmed, may offer new perspectives on the understanding of the role of RNA-protein interactions in another example of gene expression regulation. In conclusion, the committee believes that the previous solid experience in the study of RNP structure and function in vitro, combined with a more recent interest in other relevant RNP-mediated cellular processes and especially their in vivo studies, will allow the unit to give important contributions to the understanding of the molecular mechanisms underlying several circuitries of post-transcriptional control. 5 Appreciation of resources and of the life of the research unit The unit has a good scientific spirit. Many topics involve several accomplished researchers and this appears to work smoothly. From the discussion with the students, it appears that they generally considered that mentoring was overall very good. 6 Recommendations and advice Strengths : The committee was impressed by the level of involvement of the units and its members in teaching activities and in the commitment of its senior members in administration of research, both locally and at the national level, while maintaining highly dynamic and internationally visible research activities. The committee appreciated the dynamism of the groups that can be witnessed by the emergence of new research topics, in particular on the in vivo studies on new RNPs. Weaknesses : While the witnessed emergence of new research topics animated by young scientists is obviously very positive, attention should be paid in the longer term to the potential risk of dispersion and it might be important during the next few years to concentrate on the most promising new lines of research. Since the director of the unit may be renewed for the very last time, the unit has to make considerable efforts to identify the individual(s) who will be the best positioned to be the future head(s) of the unit(s). It is in the interest of every one in the unit to think in the most constructive way about the future organization of the unit and it would be best if the choices could be done collectively. 7